For details on NEB's quality controls for DNA ligases, visit our Ligase Quality page.
Figure 1: T4 DNA Ligase competitor study – nuclease contamination T4 DNA Ligase from multiple suppliers was tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3’overhang or 5’ overhang containing oligonucleotides. The percent degradation by contaminating nucleases is determined by capillary electrophoresis and peak analysis. The resolution is at the single nucleotide level.
Highlights
Isolated from a recombinant source
Supplied with 10X Reaction Buffer
Product Source
An E. coli strain that carries the cloned T4 DNA Ligase gene.
This product is related to the following categories:
One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer.
Concentration: 400,000 cohesive end units/ml and 2,000,000 cohesive end units/ml
Reaction Conditions
1X T4 DNA Ligase Reaction Buffer
Incubate at 16°C
1X T4 DNA Ligase Reaction Buffer
50 mM Tris-HCl
10 mM MgCl2
1 mM ATP
10 mM DTT
(pH 7.5 @ 25°C)
Storage Buffer
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C
ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
To dilute T4 DNA Ligase that will subsequently be stored at -20°C, 50% glycerol storage buffer (Diluent Buffer A (with rAlbumin), NEB #B8532S) should be used; to dilute for immediate use, 1X T4 DNA Ligase Reaction Buffer can be used.
Ligation can also be performed in any of the four restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer if they are supplemented with 1 mM ATP.
Room Temperature Ligation For convenience, ligations may be done at room temperature (20-25°C). For cohesive (sticky) ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 10 minutes. For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 2 hours or 1 µl high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB's Quick Ligation Kit (#M2200S) [30 reactions] or (#M2200L) [150 reactions]) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature.
References
Engler, M.J. and Richardson, C.C. (1982). P.D. Boyer(Ed.), 5, 3. San Diego: Academic Press.
Remaut, E., Tsao, H. and Fiers, W. (1983). Gene. 22, 103-113.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd Ed.). 1.53-1.73.
Sexton T, Kurukuti S, Mitchell JA, Umlauf D, Nagano T, Fraser P (2012) Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip Nat Protoc; 7(7), 1335-50. PubMedID: 22722369, DOI: 10.1038/nprot.2012.071
Publications
Thuronyi, B.W., Koblan, L.W., Levy, J.M. et al (2019) Continuous evolution of base editors with expanded target compatibility and improved activity Nat Biotechnol; 37, 1070-1079. PubMedID: 31332326, DOI: 10.1038/s41587-019-0193-0
Quality, Safety & Legal
Quality Control Assays
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
Now available as a Glycerol-free reagent. Learn more
Now available as a GMP-grade reagent. Learn more
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